Soil Biology & Biochemistry 40 (2008) 2762–2770 Contents lists available at ScienceDirect Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio Tag-encoded pyrosequencing analysis of bacterial diversity in a single soil type as affected by management and land use V. Acosta-Martı´nez a,*,S.Dowdb,Y.Sunc, V. Allen d a USDA-ARS, Cropping Systems Research Laboratory, Wind Erosion and Water Conservation Unit, Lubbock, TX 79415, USA b USDA-ARS, Cropping Systems Research Laboratory, Livestock Issues Unit, Lubbock, TX 79403, USA c Medical Biofilm Research Institute, Lubbock, TX 79410, USA d Paul Whitfield Horn Professor, Thornton Distinguished Chair at Department of Plant & Soil Science, Texas Tech University, Lubbock 79409, USA article info abstract Article history: Impacts of management and land use on soil bacterial diversity have not been well documented. Here we Received 16 May 2008 present the application of the bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) diversity Received in revised form 15 July 2008 method, which will promote studies in soil microbiomes. Using this modified FLX pyrosequencing Accepted 23 July 2008 approach we evaluated bacterial diversity of a soil (Pullman soil; fine, mixed, thermic Torrertic Paleus- Available online 24 August 2008 tolls) with 38% clay and 34% sand (0–5 cm) under four systems. Two non-disturbed grass systems were evaluated including a pasture monoculture (Bothriochloa bladhii (Retz) S.T. Blake) [P] and a diverse Keywords: mixture of grasses in the Conservation Reserve Program (CRP). Two agricultural systems were evaluated Soil management Bacterial diversity including a cotton (Gossypium hirsutum L.) -winter wheat (Triticum aestivum L.)-corn (Zea mays L.) 454 Pyrosequencing rotation [Ct–W–Cr] and the typical practice of the region, which is continuous monoculture cotton Conservation Reserve Program (Ct–Ct). Differences due to land use and management were observed in soil microbial biomass C Land use (CRP > P ¼ Ct–W–Cr > Ct–Ct). Using three estimators of diversity, the maximum number of unique Cotton sequences operational taxonomic units (OTU; roughly corresponding to the species level) never exceeded Integrated crop-livestock production 4500 in these soils at the 3% dissimilarity level. The following trend was found using the most common estimators of bacterial diversity: Ct–W–Cr > P ¼ CRP > Ct–Ct. Predominant phyla in this soil were Acti- nobacteria, Bacteriodetes and Fermicutes. Bacteriodetes were more predominant in soil under agricultural systems (Ct–W–Cr and Ct–Ct) compared to the same soil under non-disturbed grass systems (P and CRP). The opposite trend was found for the Actinobacteria, which were more predominant under non- disturbed grass systems (P and CRP). Higher GÀ bacteria and lower Gþ bacteria were found under Ct–W– Cr rotation and highest abundance of actinomycetes under CRP. The bTEFAP technique proved to be a powerful method to characterize the bacterial diversity of the soil studied under different management and land use in terms not only on the presence or absence, but also in terms of distribution. Published by Elsevier Ltd. 1. Introduction are readily cultivated (Nocker et al., 2007). It is acknowledged that molecular approaches based on PCR can also introduce their own Bacteria are the most abundant and diverse group of soil forms of bias (Farrelly et al., 1995; Suzuki and Giovannoni, 1996; organisms (Vestal and White, 1989; Gans et al., 2005). There has von et al., 1997). Also, similar to culturing, primary drawbacks to been an emerging foundation of data on bacteria found in soil as the use of molecular methods include intense labor requirements molecular methodologies, which have developed over the past and expense. In spite of this, molecular methods provide the most decade, have enhanced the ability of researchers to examine their powerful tools available for elucidation of bacterial diversity in diversity in different soils. Despite the key role of bacteria in soil environmental samples. The use of pyrosequencing for evaluating processes, there is still lack of information about the bacterial microbial diversity has been described recently as promising to be diversity of soils as affected by management and land uses. It has less labor intensive (Cristea-Fernstrom et al., 2007; Huse et al., become well known that culture-based methods are extremely 2007; Liu et al., 2007; Roesch et al., 2007; Sundquist et al., 2007; biased in their evaluation of microbial diversity, extremely time- Dowd et al., 2008a). Pyrosequencing is a DNA sequencing technique consuming, and provide information only on the 1% of bacteria that that is based on detection of released pyrophosphate (PPi) during DNA synthesis (Ronaghi, 2001). The released PPi is subsequently * Corresponding author. Tel.: þ1 806 723 5233; fax: þ1 806 723 5272. converted to ATP by ATP sulfurylase, which provides the energy to E-mail address: [email protected] (V. Acosta-Martı´nez). luciferase to oxidize luciferin and generate light proportional to the 0038-0717/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.soilbio.2008.07.022 V. Acosta-Martı´nez et al. / Soil Biology & Biochemistry 40 (2008) 2762–2770 2763 number of incorporated nucleotides. By performing hundreds of CRP, P, Ct–W–Cr and Ct–Ct. In addition, three soil samples were thousands of these reactions in parallel, the sequence of the taken (with 50 m of distance between them) within each of the template can be determined because the added nucleotides are three sites representing a system (n ¼ 9). However, DNA was known (Ronaghi, 2001). Previously, Roesch et al. (2007) enumer- extracted from one soil sample for each of the three sites repre- ated and contrasted microbial diversity of four soils from the senting a system (providing triplicates). western hemisphere by using pyrosequencing. Here we utilized Prior to conversion to CRP, P and Ct–W–Cr rotations, soils of this a modification of pyrosequencing that greatly reduces the sample region were primarily under continuous monoculture of cotton cost and allows characterization of the bacterial diversity of soil as since 1940. A detail description of the size of the sites, crop varieties affected by management and land use, which will promote studies and management applied on these systems are described in the in microbial diversity. Texas Alliance for Water Conservation report (TAWC, 2007). In Partial ribosomal amplification and pyrosequencing (PRAPS) is brief, the CRP sites were non-disturbed and non-irrigated for at a recent and powerful method which can be used to look in depth least 10 years. These sites were established under a diverse mixture at microbial diversity in virtually any type of sample (Dowd et al., of grasses typical of this region [i.e., ‘WW-Spar’ old world bluestem 2008a) using 454 Genome Sequencer FLX System (Roche, Nutley, [Bothriochloa ischaemum (L.) Keng. var. ischaemum (Hack.)], blue New Jersey). The bacterial tag encoded FLX amplicon pyrose- grama [Bouteloua gracilis (Willd. Ex Kunth) Lag. Ex Griffiths], and quencing (bTEFAP) method (Dowd et al., in press) improves upon green sprangletop (Leptochloa dubia (Kunth) Nees)]. The pasture this PRAPS approach increasing the cost-benefit of diversity pyro- sites were established with ‘WW-B. Dahl old world bluestem grass sequencing through the use of sample-specific sequence tags [Bothriochloa bladhii (Retz) S.T. Blake] under center pivot irrigation incorporated onto secondary amplification primers. Amplicons for at least 3 years. The agricultural sites under Ct–Ct and Ct–W–Cr from individual samples can then be pooled prior to 454 were conventionally tilled every autumn to 15 cm depth. Contin- sequencing and using bioinformatic approaches they can be iden- uous cotton (Ct–Ct) sites were non-irrigated (dryland) and cotton tified post-sequencing and analyzed separately. The use of 454 FLX was planted at 101.6 cm row spacing. The Ct–W–Cr rotation sites pyrosequencing is revolutionary because it can provide a sufficient were irrigated under center pivot. For this rotation, cotton was number of sequences of adequate length to enable extrapolations planted on 76.2 cm row spacing, corn was generally grown from that estimate bacterial diversity based on its two components: (1) April to August with 50.8 cm row spacing, and winter wheat was the total number of species present (species richness or abun- planted in autum. Cotton was generally planted during May every dance), and (2) the distribution of individuals among those species year in Ct–Ct and Ct–W–Cr systems. The agricultural sites (Ct–Ct (species evenness or equitability; Margalef, 1958; Kennedy, 1999). and Ct–W–Cr) have been under the management described for at Thus, 454 FLX pyrosequencing can provide more meaningful least 3 years. All irrigated sites have received between 127 and comparisons of bacterial diversity of soil as affected by manage- 508 mm of water depending on the year. ment and land use (Roesch et al., 2007). Understanding soil microbial community structure shifts 2.2. Selected soil properties following implementation of various land use and management systems is an important component in selecting management Soil pH was determined on air-dried subsamples (sieved to practices to improve ecosystem services and soil functions. Here we <5 mm) using a glass combination electrode with a soil:water ratio utilized the bTEFAP to evaluate bacterial diversity in the same soil of 1:2.5. Soil total C and N contents were determined in subsamples under two undisturbed grass systems that included Conservation of air-dried soil (sieved to <180 mm) by automated dry combustion Reserve Program (CRP), which had been undisturbed for 15 years, (LECO TruSpec CN) in a private laboratory (Ward Laboratories, and pasture (P), which has been undisturbed for 3 years, and two Nebraska, USA). The soil microbial biomass C (MBC) and microbial agricultural systems including a cotton–wheat–corn (Ct–W–Cr) biomass N (MBN) were determined on a 15-g oven-dry equivalent rotation and continuous monoculture cotton (Ct–Ct).